The need is pressing to develop an effective and safe vaccine against the human immunodeficiency virus (HIV) with the primary objective of arresting the spread of the AIDS epidemic. Design of such efficacious vaccines will be facilitated by the elucidation of immune correlates of vaccine-induced protection. We have conducted vaccination studies in rhesus macaques to evaluate a plasmid DNA containing a vif-deleted SIVmac239 provirus (SIVvif provirus) as a proviral DNA vaccine. Furthermore we investigated the adjuvant activity of a rhesus macaque interleukin (IL)-15 expression plasmid when co-inoculated with the SIV?vif proviral DNA. Findings from these studies indicated that co-immunization with an IL-15 plasmid expression vector affords a significant adjuvant effect to the SIV vif-deleted proviral DNA vaccine and enhanced protection against vaginal challenge with pathogenic SIVmac251. Furthermore, examination of SIV-specific cellular immune responses by interferon-? ELISpot and T cell proliferation assays revealed a significant enhancement of interferon-? ELISpot responses by inclusion of IL-15 as an adjuvant for the proviral DNA vaccine. These NIH-funded vaccine studies have now been concluded. We propose to further examine virus-specific cellular immune responses from cryopreserved PBMC samples banked from these same vaccine studies, using a 10 color multi-parameter flow cytometric assay. This assay will evaluate SIV-induced T cell intracellular expression of cytokines including interferon-?, tumor necrosis factor (TNF)-a, and interleukin-2 and also test for expression of specific cell surface markers for cytotoxic T cell degranulation (CD107a), CD8 T cell memory development (CD127 or IL-7 receptor a) and a negative immunoregulatory protein (program death 1/PD-1). This type of immune response assessment was not described in the original NIH proposal that funded these SIV?vif DNA vaccine studies. However, this investigation is now well justified by observations from these studies and by recent reports showing the utility of these functional profiles to elucidate immune correlates that are critical for vaccine design. The hypothesis is that implementation of novel tools for comprehensive immune response analysis in macaques vaccinated with SIV?vif proviral DNA with or without IL-15 expression plasmid, will elucidate an immune correlate(s) for IL-15 adjuvant activity and vaccine-induced control of virus load. Furthermore, Il-15 has been considered as a potential immunotherapeutic for patients on HAART. Accordingly, careful elucidation of cellular immune responses associated with a potentially effective cytokine adjuvant such as IL-15 may impact design of future HIV-1 vaccines and therapeutics. PUBLIC HEALTH RELEVANCE: Findings from recent studies in monkey animal models, suggested that including a lymphocyte growth factor designated interleukin (IL)-15, as part of the highly attenuated SIV?vif DNA vaccine, improved the effectiveness of this vaccine. However standard tests for vaccine-induced immune responses did not identify a specific cause or mechanism by which IL-15 improved the vaccine. We propose to further examine virus-specific cellular immune responses using new technologies developed over the past decade, to identify mechanisms by which IL-15 improved this DNA vaccine. Findings from these studies may then be used for future design for more effective HIV-1 vaccines, as well as design for immunotherapeutics for HIV-1 infection. [unreadable] [unreadable] [unreadable]